Small cell lung cancer (SCLC) has an exceptionally high metastatic potential, and the majority of patients have extensive stage disease at the time of diagnosis. While most patients are highly responsive to chemotherapy, disease recurrence is universal, and recurrent disease is largely unresponsive to therapy. The molecular basis for the dramatic shift from a highly chemosensitive disease at diagnosis to a highly chemorefractory and lethal disease a few months later has not been defined. A key contributor to our lack of knowledge about this tumor evolution is that SCLC is rarely re-sampled at the time of disease progression. This project will take complementary approaches, using next generation sequencing to comprehensively characterize changes associated with acquired therapeutic resistance in human SCLC. The first Aim is based on analysis of acquired resistance in patient-derived xenograft (PDX) models. We have generated a large library of PDX lines from patients with newly diagnosed, treatment-nave SCLC. With repeated exposure of tumor-bearing mice to standard first line chemotherapy, we have generated chemoresistant derivatives, precisely as occurs in the clinic. We will use pair-wise analysis to assess recurrent genomic and epigenetic changes associated with acquired chemoresistance in SCLC. The second Aim is based on analysis of circulating tumor cells (CTC) from patients with SCLC. CTC will be used as a source of tumor genomic DNA through which we can similarly assess genomic and epigenetic changes associated with acquired resistance, linking the results found in Aim 1 to the clinic. The third Ai will validate lead candidate drivers of acquired resistance in SCLC identified in the first 2 Aims. Candidates will be validated by a series of complementary approaches both in vitro and in vivo, including use of novel approaches to targeted modification of gene expression in the PDX models described in Aim 1. Data obtained in this study will define mechanisms of acquired resistance in SCLC and will provide insight into the relative merits of invasive tumor biopsy vs. CTC collection as sources for comprehensive tumor mutational profiling. These data will influence clinical research strategies for the treatment of SCLC, and have broad-based implications for acquired chemotherapeutic resistance in other diseases.